Orientadores: Anibal Eugenio Vercesi, Karina Gottardello Zecchin / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-15T13:27:40Z (GMT). No. of bitstreams: 1 Rossato_FrancoAparecido_M.pdf: 961339 bytes, checksum: eb647604d3856b3e12d36a8143d26540 (MD5) Previous issue date: 2010 / Resumo: Ácido graxo sintase (FASN - EC 2.3.1.85) é a enzima responsável pela síntese endógena de ácidos graxos de cadeia longa a partir dos precursores acetil-CoA e malonil-CoA. Diversos estudos mostram que a FASN é altamente expressa em vários tipos de neoplasias malignas humanas, tais como de próstata, mama, melanoma e, em alguns destes tumores, a alta expressão de FASN está associada a um pior prognóstico. O tratamento com inibidores específicos de FASN, como cerulenina, C75 e orlistat, diminui a capacidade de proliferação e induz apoptose em linhagens celulares derivadas de neoplasias malignas de próstata, mama e cólon, porém pouco se sabe sobre os efeitos desses inibidores em células não tumorais. Recentemente mostramos que a inibição de FASN com orlistat reduz a proliferação e induz apoptose em células B16-F10 de melanoma murino (Carvalho et al. 2008). Considerando que (1) pouco é conhecido sobre os efeitos de inibidores de FASN em células "normais", inclusive melanócitos e (2) dados iniciais mostram que o tratamento com orlistat ou cerulenina também induz elevados níveis de apoptose em células "normais", este estudo teve por objetivo principal verificar os mecanismos envolvidos na morte induzida pela inibição da FASN em linhagem celular não-tumorigênica derivada de melanoblastos de camundongos (melan-a). O tratamento in vitro de células melan-a com 5 µg/mL de cerulenina ou com 30 µM de orlistat induziu expressiva porcentagem de apoptose, mas não necrose. As células tratadas também apresentaram redução da proliferação, além de discretas ativação de caspase-3 e liberação de citocromo c. Como o silenciamento de FASN através de RNA de interferência (RNAi) não resultou em apoptose, investigamos o possível envolvimento mitocondrial na morte induzida pelos inibidores de FASN. De fato, o tratamento com cerulenina ou orlistat resultou em diminuição do ??m, além de mais de 50% de inibição da velocidade de respiração das melana no estado de repouso. Paralelamente também foi constatado que esses mesmos inibidores de FASN induzem apoptose e reduzem a proliferação de células derivadas de queratinócitos não tumorigênicos, HaCaT. O presente trabalho mostra, portanto, que os inibidores de FASN, cerulenina e orlistat, apresentam efeitos nocivos sobre células não tumorais, conseqüência da ação sobre a respiração mitocondrial. / Abstract: Fatty acid synthase (FASN - EC 2.3.1.85) is the enzyme responsible for endogenous synthesis of long chain fatty acid palmitate derivate from precursors acetyl-CoA and malonyl-CoA. Studies have shown that FASN is highly expressed in several types of human malignancies, such as prostate, breast, melanoma, and in some of these tumors, high expression of FASN is associated with a poor prognosis. FASN inhibitors, such as cerulenin, C75, and orlistat, decrease cell proliferation and induce apoptosis in prostate, breast, and colon tumor cells lines. Recently we demonstrated that inhibition of FASN with orlistat reduced proliferation and induced apoptosis in cells B16-F10 murine melanoma (Carvalho et al. 2008). Consider that (1) little is known about the effects of FASN inhibitors in normal cells, including melanocytes and (2) previous data show that treatment with orlistat or cerulenin also induces high levels of apoptosis in normal cells, the aim of this study was to analyze the mechanisms involved in FASN inhibitioninduced cell death in cell line derived from non-tumorigenic mice melanoblasts (melana). In vitro treatment of melan-a cells with 5 µg/mL cerulenin or 30 µM orlistat induced a significant percentage of apoptosis, but not necrosis. Treated cells also showed reduced proliferation, and moderate activation of caspase-3 and release of cytochrome c. As FASN silencing through RNA interference (RNAi) did not result in apoptosis, we investigated the possible involvement of mitochondria in FASN inhibition-induced cell death. Cerulenin or orlistat treatment of melan-a cells decreased ?? m and inhibited more than 50% the respiration rate in rest state. We also detected significant apoptosis and reduced proliferation in cells derived from non-tumorigenic keratinocyte, HaCaT, after incubation with the same FASN inhibitors. In conclusion, this study shows that FASN inhibitors, cerulenin and orlistat, have adverse effects on non-tumor cells, as a consequence of direct action on mitochondrial respiration. / Mestrado / Biologia Estrutural, Celular, Molecular e do Desenvolvimento / Mestre em Fisiopatologia Médica

Orientadores: Anibal Eugênio Vercesi, Karina Gottardelo Zecchin / Texto em português e inglês / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-24T16:40:04Z (GMT). No. of bitstreams: 1 Rossato_FrancoAparecido_D.pdf: 7526681 bytes, checksum: 3768c017da7bde02ddd95e8e41881438 (MD5) Previous issue date: 2014 / Resumo: Recentemente mostramos que os inibidores da enzima ácido graxo sintase (FASN - EC 2.3.1.85), cerulenina e orlistat, reduzem a proliferação e induzem apoptose em células B16-F10 de melanoma murino via mecanismos mitocondriais. Neste presente estudo investigamos os efeitos desses inibidores de FASN em linhagem celular não-tumorigênica derivada de melanoblastos de camundongos (melan-a). O tratamento in vitro de células melan-a com 5 µg/mL de cerulenina ou com 30 µM de orlistat inibiu a proliferação celular, com acúmulo da proteína supressora de tumor p21WAF1/Cip1, assim como induziu a via intrínseca da apoptose com liberação de citocromo c e ativação de caspases-3 e -9, sem ativação da caspase-8. Os inibidores de FASN não alteram o conteúdo de ácidos graxos livres nas células melan-a, verificados por espectrometria de massas, sugerindo que o tratamento com cerulenina ou orlistat induz apoptose independente da inibição desta enzima. Análise das funções da bioenergética mitocondrial das células melan-a mostraram inibição da respiração, seguido por aumento da produção de superóxido. A inibição da respiração, promovida pelo tratamento com cerulenina ou orlistat, foi restrita à oxidação de substratos ligados a NADH (39,9% DMSO x cerulenina; ou 60,8% EtOH x orlistat) e succinato (45,8% DMSO x cerulenina; ou 51,8% EtOH x orlistat), e não foi significativa quando as mitocôndrias estavam respirando com substrato do complexo IV, N,N,N',N'-tetrametil-p-fenilenodiamina. A proteção conferida pelo sequestrador de radicais livres N-acetil cisteína (NAC) sugere que a disfunção mitocondrial provocada por estes compostos está associada a estresse oxidativo e é provável que seja mediada pela ação de superóxido na cadeia respiratória nos níveis de complexos de I e II. Análise proteômica de mitocôndria dessas células também mostra alterações ligadas ao estresse oxidativo. Nossos dados em conjunto sugerem que cerulenina e orlistat induzem apoptose em células não tumorais como resultado de uma disfunção mitocondrial e de maneira independente de FASN / Abstract: We have previously reported that the fatty acid synthase (FASN) inhibitors, cerulenin or orlistat, induce apoptosis in B16-F10 mouse melanoma cells mediated by mitochondria. Here we investigate the effects of these inhibitors on the non-tumorigenic mouse cell line melan-a. Cerulenin or orlistat treatment decreased cells proliferation, accompanied by increased amounts of the tumor suppressor protein p21WAF1/Cip1, as well as induced apoptosis, but not necrosis, in melan-a cell line. Mitochondrial cytochrome c release and activation of caspases-9 and -3 were detected in melan-a-treated cells. siRNAi for FASN did not culminate in apoptosis, and FASN inhibitors treatment did not alter free fatty acids content in the non-tumorigenic cells, as verified by mass spectrometry, suggesting that cerulenin or orlistat induces apoptosis independent on FASN inhibition. Analysis of energy-linked functions of melan-a mitochondria showed inhibition of respiration followed by large stimulation of superoxide production. Respiratory inhibition after cerulenin or orlistat treatment, respectively, was restricted to the oxidation of NADH-linked substrates (39.9 or 60,8%) and succinate (45.8 or 51.8%) and was not significant when mitochondria were respiring on the complex IV substrate, N,N,N?,N?-tetramethyl-p-phenylendiamine. The protection conferred by the free radical scavenger NAC suggests that the mitochondrial dysfunction caused by these compounds is associated with oxidative stress and is mediated by the action of superoxide on the respiratory chain at the levels of complexes-I and II. Proteomic analysis of mitochondria melan-a cells also indicate major changes linked to oxidative stress. Taken together, the present results show that cerulenin or orlistat induces apoptosis in non-tumorigenic cells via mitochondrial dysfunction, independent on FASN inhibition / Doutorado / Fisiopatologia Médica / Doutor em Ciências

Le but de cette thèse est d’étudier les modifications biochimiques mitochondriales liées à un défaut de lipoylation des protéines dans les fibroblastes de 14 patients. Ces patients sont porteurs d’une mutation dans un gène codant une des protéines impliquées soit dans la synthèse de l’acide lipoïque (LIPT1, LIPT2) soit dans la voie de biogenèse des centres Fe-S mitochondriale (FDX1L, ISCA1, ISCA2, IBA57, NFU1, BOLA3). La voie de biogenèse des centres Fe-S est nécessaire à la maturation des protéines Fe-S mitochondriales, dont la lipoic acid synthase (LIAS).Ces travaux ont permis d’étudier notamment un deuxième cas de déficit en FDX1L ainsi qu’un patient porteur d’une nouvelle mutation dans ISCA1. Les déficits dans la voie de la biogenèse des centres Fe-S observés chez les patients étudiés affectent principalement la maturation des protéines mitochondriales à centre [4Fe-4S] dont l’aconitase mitochondriale, les complexes I et II de la chaîne respiratoire et la LIAS, induisant ainsi un défaut de lipoylation d’enzymes clés du métabolisme énergétique (PDHc, KGDHc). Aucune atteinte du réseau mitochondrial ni de variations du stress oxydatif n’ont pu être mises en évidence. Finalement, l’ajout d’acide lipoïque exogène n’améliore pas les déficits observés.Les profils d’expression des protéines dans les fibroblastes des patients suggèrent que les protéines NFU1, BOLA3 et IBA57 ainsi que ISCA1, ISCA2 et IBA57 coopèrent entre elles de manière complexe. / The aim of this work is to study mitochondrial dysfunctions related to a defect of protein lipoylation in fibroblasts of 14 patients. These patients carry a point mutation in a gene encoding for a protein involved either in lipoic acid biosynthesis (LIPT1 or LIPT2) or in the mitochondrial pathway devoted to iron-sulfur cluster biogenesis (FDX1L, ISCA1, ISCA2, IBA57, NFU1, BOLA3) essential for maturation of mitochondrial Fe-S proteins such as lipoic acid synthase (LIAS). This work describes the second case of FDX1L deficiency and a patient with a new mutation in ISCA1 gene.We found that mitochondrial [4Fe-4S] proteins (mitochondrial aconitase, complexes I and II of the respiratory chain and LIAS) are mainly affected in fibroblasts of patients with defect in the mitochondrial Fe-S maturation pathway. Secondary, LIAS dysfunction leads to decreased lipoylation of PDHc and KGDHc, complexes involved in energy metabolism. Neither mitochondrial network nor oxidative stress biomarkers was modified in our study. Addition of exogenous lipoic acid did not rescue the mitochondrial deficiency.Protein expression profiles obtained in fibroblasts of patients suggest that NFU1, BOLA3 and IBA57 and also ISCA1, ISCA2 and IBA57 could function and interact together to form protein complexes.

Au laboratoire, une famille de tyrosine kinases propres aux bactéries et ne présentant aucune ressemblance structurale avec les protéine-kinases d’origine eucaryote a été identifiée. Ces enzymes, appelées BY-kinases, sont notamment impliquées dans la biosynthèse des polysaccharides extracellulaires, mais leurs rôles précis ainsi que leurs mécanismes catalytiques sont encore peu compris.Dans la première partie de ce travail, nous avons caractérisé le rôle physiologique de la phosphorylation sur la tyrosine de la protéine Ugd, une UDP-glucose déshydrogénase, par les BY-kinases Wzc et Etk d’E. coli. Nous avons démontré que la phosphorylation d’Ugd sur un site commun à Wzc et Etk augmente son activité. Nous avons également établi que la phosphorylation d’Ugd par Wzc participe à la régulation de la quantité d’acide colanique produit, tandis que la phosphorylation d’Ugd par Etk influence la résistance de la bactérie à la polymyxine.Nous avons également effectué une analyse structure-fonction du domaine cytoplasmique de deux BY-kinases, CapA1/CapB2 de S. aureus et Wzc d’E. coli. Nous avons montré que ces deux protéines s’associent en octamère, grâce au motif EX2RX2R et qu’elle s’autophosphoryle selon un mécanisme intermoléculaire. Nous avons, de plus, identifié le mécanisme d’activation de ces protéines et révélé l’importance d’un domaine particulier dans l’autophosphorylation de Wzc et la biosynthèse de l’acide colanique.La caractérisation structurale et fonctionnelle des BY-kinases représente une approche prometteuse et originale en vue de l’élaboration de molécules inhibant spécifiquement leur activité et pouvant affecter le pouvoir virulent des bactéries pathogènes. / A new class of bacterial enzymes, named BY-kinases, has been shown to catalyze protein-tyrosine phosphorylation. These enzymes share no structural and functional similarities with their eukaryotic counterparts. Evidence of their involvement in extracellular polysaccharide biosynthesis has been provided, but their accurate functions and their catalytic mechanism remain largely unknown.First, we characterized the physiological role of tyrosine phosphorylation of Ugd, a UDP-glucose dehydrogenase, by the BY-kinases Wzc and Etk of E. coli. We demonstrated that Ugd phosphorylation by Wzc or Etk occurs on the same site and increases its activity. We also established that Wzc-mediated phosphorylation of Ugd participates in the regulation of colanic acid production whereas Ugd phosphorylation by Etk influences resistance to polymyxin.In addition, we performed a structure-function analysis of the cytoplasmic domain of two BY-kinases, namely CapA1/CapB2 from S. aureus and Wzc from E. coli. We showed that these two proteins associate in a ring-shaped octamer in which the motif EX2RX2R plays a crucial role. In addition, we showed that BY-kinases autophosphorylate using an intermolecular mechanism. We also identified the activation mechanism of BY-kinases and we revealed the role of a particular domain, found specifically in BY-kinases from proteobacteria, in Wzc autophosphorylation and colanic acid biosynthesis.Structural and functional characterization of BY-kinases represents an original and promising approach in order to develop new molecules inhibiting specifically these enzymes and to affect the virulence of bacterial pathogens.

This thesis reports diverse synthetic and mechanistic studies in six chapters, as summarized below. Chapter 1. Revised mechanism and improved methodology for the perkin condensation.1 The generally accepted mechanism for the well-known Perkin condensation is unviable for at least two reasons: (1) the normally employed base, acetate ion, is too weak to deprotonate acetic anhydride (Ac2O, the substrate); and (2) even were Ac2O to be derprotonated , its anion would rapidly fragment to ketene and acetate ion at the high temperatures employed for the reaction. It has proved in this study that the Perkin condensation occurs most likely via the initial formation of a fem-diacetate (3, Scheme 1) from benzaldehyde (2) and acetic anhydride (1).1 The key nucleophile appears to be the enolate of 3 (and not of 1), which adds t the C=O group of the aldehyde 2 (present in equilibrium with 3). Thus cinnamic acid (4a) was formed in -75% yield with 3 as the substrate under the normal conditions of the Perkin reaction. The deprotonation of the diacetate appears to be electrophilically assisted by the neighbouring acetate group, the resulting enolate being also thermodynamically stabilized in form of an orthoester (I). The possibility that the diacetate 3 is the actual substrate in the Perkin reaction indicates that the reaction can be effected under far milder conditions, with a base much stronger than acetate ion. This was indeed realized with potassium t-butoxide in dioxane, which converted the gem-diacetates derived from a variety of aromatic aldehydes to the corresponding cinnamic acids (4), rapidly and in good yields at room temperature (Scheme 2). This represents a vast improvement in the synthetic protocol for the classical Perkin reaction, which remains an important carbon-carbon bond forming reaction to this day. Chapter 2. Aromaticity in azlactone anions and its sifnificance for the Erlenmeyer synthesis.2 The classical Erlenmeyer azlactone synthesis of amino acids occur via the formation of an intermediate azlactone, and its subsequent deprotonation by a relatively weak base(acetate ion),. The resulting azlactone anion (cf. II, Scheme 3) functions as a glycine enolate equilvalent, and is considered in situ with an aromatic aldehyde, subsequent dehydration leading to the 4-alkylidene oxazolone(analogously to the Perkin reaction). Interestingly, azlactone anions are possibly aromatic, as they possess 6π electrons in cyclic conjugation; this would explain their facile formation as also the overall success of the Erlenmeyer synthesis. The following studies evidence this possibility. The strategy involved studying the rates of base-catalyzed deprotonation in 2-phenyl-5(4H)-oxazolone (azlactone, 5) and its amide and ketone analogs, 3-methyl-2-phenyl-4(5H)-imidazolone (6), and 3,3-dimethyl-2-phenyl-493H)-pyrrolone (7) respectively.2 Two processes were studied, deuterium exchange and condensation with hexadeuteroacetone (Scheme3): both are presumably mediated by the anions II-IV, so their stabilities would govern the overall rates. These were followed by 1H NMR spectroscopy by monitoroing the disappearance of the resonance of the proton α to the carbonyl group. The order of deprotonation was found to be 6 > 5 > 7. However, the expected order based on pKa values would be ketone > ester > amide, i.e. 7 > 5 > 6. The inverted order observed strongly indicates the incursion of aromaticity, which would be enhanced by the electron-donor capabilities of the heteroatoms is 5 and 6. This is further substantiated by the greater reactivity in the case of the nitrogen analog 6 relative to the oxygen 5, which parallel the electronegativity order. (The aromaticity order would thus be: III > II > IV. The imidazole nucleus is indeed to be considerably more aromatic than the oxazole.) The synthesis of the analogs 6 and 7 was accomplished via an interesting intramolecular aza-Wittig reaction (Schemes 4 & 5) Chapter 3. Umpolung approach to the Erlenmeyer process in the synthesis of dehydro amino acids. These studies are based on the general observation that most of the strategies for the synthesis of α-amino acids introduce the side chain (or part was inverted in an umpolung sense. The key reaction studied was that of 2-phenyl-4-ethoxymethylne-5(4H)-oxazolone (11) with Grignard reagents: this resulted in the opening to yield a protected dehydro amino acid (12), in good to excellent yields (65-87%)(Scheme ^). As the azlactone reactant 11 is the ekectrophilic partner, this may be viewed as a partial umpolung version of the classical Erlenmeyer process. The readily available reactants, simple procedure and mild reaction conditions make this a very attractive method for the synthesis of a variety of α-dehydro amino acids. Chapter 4. The Erlenmeyer azlactone synthesis with aliphatic aldehydes under solvent-free microwave conditions. 3 A serious limitation to the classical Erlenmeyer reaction is that it generally fails in the case of aliphatic aldehydes. This chapter describes a convenient approach to this problem that extends the scope of the Erlenmeyer synthesis, via a novel microwave-induced, solvent-free process. This, it was observed that azlactones (5) react with aliphatic aldehydes (13) upon adsorption on neutral alumina and irradiation with microwaves (< 2 min), forming the corresponding Erlenmeyer products (14) in good yields (62-78%, Scheme 7). (The possible mechanistic basis of the procedure, which is presumably mediated by V , is discussed).3 Chapter 5. 2,4, 10-Trioxaadamantane as a carboxyl protecting group: application to the asymmetric synthesis of α-amino acids (umpolung approach).It is known that the 2,4,10-trioxaadamantane moiety is not only remarkably stable to nucleophilic attack, but can also be easily hydrolyzed to the corresponding carboxylic acid.4 It was of interest to apply this carboxyl protection strategy for designing a synthesis of α-amino acids, essentially by starting with a protected glyoxylic acid. The corresponding aldimine was expected to (nucleophilically) add organometallic reagents at the C=N moiety (cf. Shceme 8), the side chain of the amino acid being thus introduced in umpolung fashion. Also, a chiral aldimine would define an asymmetric synthesis of amino acids. Indeed, the chiral aldimine 17, derived from 2,4,10-troxaadamantane-3-carbaldehyde 15 and [(S)-(-)-1-phenylethylamine] 16, reacted with a variety of Grignard reagents to furnish the corresponding protected α-amino acids (18) in good yields, with moderate diastereometric excess (Scheme 8). Better yields and ‘de’ values were obtained with organolithium reagents. Chapter 6: possible one-pot oligopeptide synthesis with azlactones or amino acid N-carboxyanhydrides (NCAs). This chapter describes a novel approach to oligopeptide synthesis employing azlactones or NCA’s as amino acid equivalents which are simultaneously protected and activated (Scheme 9). Thus, the addition of the 4-substituted 2-benzyloxyazlactone (19) to an N-protected amino acid under basic conditions, was initially explored. The reaction was expected to yield a dipeptide (21) via the rearrangement of the mixed anhydride intermediate (VI) (Scheme 9). The subsequent addition of a different azlactone to the dipeptide (21) would analogously lead to the formation of a tripeptide (22). This may be performed repetitively to define a strategy for C-terminal extension of an oligopeptide chain, noting that no intervening deprotecting and activating steps are necessary. (In toto deprotection may be effected finally via the hydrogenolyis of the bvenzyloxy groups, to obtain 23.) A closely analogous strategy may also be envisaged by employing N.carboxyanhydrides (NCA’S, 24) instead of azlactones, as shown in Scheme 10 (forming dipeptide 26 and tripeptide 27). The main difference n this case is that the carbamic acid moiety of the intermediate mixed anhydride (VII) is expected to undergo decarboxylation to VIII (thus obviating the need for a deprotection step). However, this putative advantage is offset by the instability of NCA’s and their tendency toward polymerization. However, only partial success could be achieved in these attempts, although a variety of conditions were explored. The strategy and the experimental results have been analyzed in detail, as this interesting approach appears to be promising, and worth further study. (For structural formula pl refer the pdf file)

Malaria, caused by Plasmodium, is one of the most devastating infectious diseases of the world in terms of mortality as well as morbidity (WHO, 2002). The development of resistance in the Plasmodium falciparum against the present antimalarials has made the situation very alarming (Trape et al., 2000). To combat this situation, new antimalarials as well as identification of new drug targets are urgently required. The discovery of the presence of type II fatty acid biosynthesis system in the malarial parasite has offered several promising new targets for this mission. This thesis describes the successful cloning of fabZ from Plasmodium falciparum, its expression in E. coli, single step affinity purification, kinetic characterization and most importantly discovery of two small molecule inhibitors (Sharma et al., 2003). The study was executed to gain insights into the structure and function of PfFabZ to get better understanding of the interactions with its substrate analogs, novel inhibitors and also acyl carrier protein (PfACP). The molecular details of the interactions of the two novel inhibitors were also determined. Lastly, the residues of PfFabZ important for the interaction with PfACP were successfully elucidated. Chapter 1 presents a brief review of the literature about the disease as well as the life cycle, biology and the metabolic pathways operational in malarial parasite, Plasmodium falciaparum. The discovery of type II FAS in P. falciparum and the aims and the scope of the thesis are also discussed. The quest of developing new antimalarials, study of the mechanism of actions of antimalarials such as quinine and its derivatives along with the major metabolic pathways (Purine, pyrimidine, phospholipids, carbohydrate metabolism, folate and heme biosynthesis pathways etc.) existing in P. falciparum are described in detail in this chapter. Origin and importance of apicoplast in P. falciaprum is also described in brief. For long, it was believed that Plasmodium spp. are incapable of de novo fatty acid synthesis but this view has undergone substantial revision due to the recent discovery of plant and bacterial type of fatty acid biosynthesis pathway in them (Surolia and Surolia, 2001). As this pathway is distinct from that of the human host it has accelerated the momentum for the discovery of new antimalarials (Surolia and Surolia, 2001). The Chapter also surveys the details of type II FAS in bacteria, particularly that of E. coli (Rock and Cronan, 1996). The dehydratase step which is the third step of fatty acid elongation cycle has been covered in considerable detail. Lastly, it focuses on the recent advancement in the understanding of fatty acid biosynthesis system in Plasmodium falciparum along with some inhibitors targeting the malarial FAS. As each enzyme of the Plasmodium FAS can serve as good antimalarial targets, my work focuses on the dehydratase step catalyzed by β-hydroxyacyl-ACP dehydratase (PfFabZ). Cloning, expression and kinetic characterization of PfFabZ forms the major content of Chapter 2. The PlasmoDB data base was searched for this gene and the mined out open reading frame contained sequence of the putative FabZ together with the bipartite leader polypeptide. Our aim was to clone the mature PfFabZ without the bipartite leader sequence. Amplification of the mature pffabZ using Plasmodium falciparum genomic DNA revealed the presence of an intron in the ORF and the gene was finally cloned by RT-PCR in pET-28a(+) vector. It was expressed with an N-terminal hexahistidine tag in BL-21(DE3) cells and purified to near homogeneity but the protein was insoluble and unstable. Truncation of 12 residues from the N-terminal end improved the stability and solubility of the protein by 3-5 fold. Truncated PfFabZ was used for all future experiments. FabZs from other sources are reported to be hexamer in solution but PfFabZ showed homodimeric arrangement in the conditions used for gel filtration as well as dynamic light scattering studies. Kinetics of PfFabZ was characterized using substrate analogs, β-hydroxybutryl-CoA (forward substrate) and Crotonoyl-CoA (reverse substrate). Both the forward and reverse reaction were thoroughly characterized by spectrophotometry and HPLC and the reverse reaction was found to be 7 times faster than the forward reaction. Km οf crotonoyl-CoA was calculated to be 86 µM and kcat/Km of 220 M-1s-1 whereas the Kmfor β-hydroxybutryl-CoA was found to be 199 µM and kcat/Kmof 80.2 M-1s-1. The kinetic data clearly indicates the higher affinity of PfFabZ for the reverse substrate. Chapter 3 describes the discovery of two small molecules inhibitors, NAS-21 and NAS-91 for PfFabZ, their detailed inhibition kinetics and their effect on the growth of Plasmodium falciparum in culture. These inhibitors were the first inhibitors to be reported for FabZ class of enzymes with an IC50 ranging below 15 µM. Both of them inhibited PfFabZ following competitive kinetics with respect to the substrates utilized for both the forward and reverse reactions. The inhibition data were analyzed by Lineweaver-Burk and Dixon plots and both inhibitors showed competitive inhibition kinetics with dissociation constant in submicromolar range. Binding constants for both the inhibitors were also determined by fluorescence titration method and were calculated to be 1.6 (± 0.04) X 106 M-1 for NAS-91 and 1.2 (± 0.03) X 106 M-1 for NAS-21. These inhibitors were checked on Plasmodium falciparum culture and both inhibited parasite growth with IC50 values of 7 µM and 100 µM for NAS-21 and NAS-91, respectively. They also inhibited the incorporation of [1,2-14C]-acetate in the fatty acids of the P. falciparum conforming the inhibition of fatty acid biosynthesis. FabZ class of enzymes are thought to contain His-Glu as a catalytic dyad. Based on the disparity in the arrangement of residues at the active site of the dimeric (Swarnamukhi et al., 2006) and hexameric forms of PfFabZ in the crystal structures (Kosteriva et al., 2005), we set out to elucidate the active site residues in PfFabZ which is described in Chapter 4. The role of each of the presumed active site residues His-133 and Glu-147 along with Arg-99 and His-98 were analyzed by chemical modification studies and site directed mutagenesis. Single and double mutants were prepared and the activity of the mutants was monitored by spectrophotometry and isothermal titration calorimetry (ITC). It was concluded that in PfFabZ, His-133 and Glu-147 makes the catalytic dyad, His-98 might be important in directing the substrate in correct orientation while Arg-99 is involved in maintaining the active site loop in proper orientation rather than taking direct part in catalysis. Chapter 4 also concludes that dimeric form of PfFabZ is inactive species and turns into active hexameric form in the presence of substrate. Chapter 5 describes the molecular details of NAS-21 and NAS-91 interactions with PfFabZ. The fact that both these compounds inhibited PfFabZ in competitive manner, prompted me to examine their interaction with the residues in the active site tunnel. Apart from the His-133 and Glu-147 catalytic dyad the only polar residue is His-98 and chemical modification and site directed mutagenesis studies were done to elucidate the interactions of these residues with NAS-21 and NAS-91. Both the inhibitors were able to protect the modification of histidines by DEPC in wild type PfFabZ, His-98-Ala mutant and His-133-Ala mutant but with differential strength, indicating that they do interact with histidines. The interaction of these inhibitors was further confirmed by determining the dissociation constants of wPfFabZ, His-98-Ala, His-133-Ala, His-98-Ala/His-133-Ala double mutant, Glu-147-Ala mutant by fluorescence titration method. The results obtained from chemical modification and fluorescence titration studies confirmed that NAS-21 interacts strongly with histidines, His-98 and His-133 but not with Glu-147. On the other hand NAS-91 interacts loosely with His-98 and His-133 but strongly with Glu-147. Chapter 5 concludes with the observation that both the inhibitors (NAS-21 and NAS-91) interact with the active site residues of PfFabZ, preventing the substrate to enter the active site tunnel. Acyl carrier protein (ACP) is a small acidic protein to which the acyl chain intermediates are tethered and shuttled from one enzyme to another for the completion of fatty acid elongation cycle. Whenever acyl carrier proteins are expressed in E. coli, they are present in three forms apo, holo and acyl-ACPs. Chapter 6 describes a novel method for the expression of histidine tagged PfACP in pure holo form, protocol for the cleavage of his-tag from PfACP by thrombin preparation of homogenous singly enriched ie PfACP [15N]-labeled or [13C]-labeled PfACP as well as doubly enriched [15N]-[13C] PfACP samples for its structure elucidation by NMR (Sharma et al., 2005). These studies also constituted reporting of a holo-ACP structure from any of the sources for the first time (Sharma, et. al. 2006). The purified pure holo-PfACP was further used for the interaction studies with PfFabZ. Earlier studies have shown that ACP interacts with FAS enzymes via helix II with conserved set of residues but the molecular details of the interactions are poorly known (Zhang, et. al., 2003). We have recently solved the NMR structure (Sharma, et. al., 2006) of PfACP and crystal structure of PfFabZ (Swarnamukhi, et. al., 2006). So, both the structures were docked using Cluspro server. Chapter 7 elucidates the roles of important residues on PfFabZ surface near the active site entry which are responsible for interacting with PfACP. The residues lining the active site entry were identified and mutated. The residues lining the active site tunnel of PfFabZ are Arg102, Lys104, Lys105, Lys123, Leu94, Phe95, Ala96, Gly97, Ile128, Ile145, Phe150 and Ala151. Charged residues were mutated to alanine and also to oppositely charged residues while the neutral residues were changed to charged residues. The interaction of PfFabZ mutants with PfACP was studied by ACP independent enzymatic assay and surface plasmon resonance (SPR) spectroscopy. It was concluded that PfFabZ and PfACP interaction is mainly governed by electrostatic interaction made by the charged residues (Lys104 being the most important residue) and is fine tuned by hydrophobic interactions. Chapter 8 summarizes the findings of the thesis. FabZ from Plasmodium falciparum was cloned and biochemically characterized. Two inhibitors for this enzyme were discovered and their molecular details of binding to PfFabZ were elucidated. The presence of catalytic dyad was confirmed and finally the residues of PfFabZ important for interaction with PfACP were elucidated. Appendix I describes the inhibition of PfENR (enoyl ACP reductase), the rate limiting and the fourth enzyme of the fatty acid elongation pathway by green tea extracts. Three tea catechins (EGCG, EGC and ECG) and two plant polyphenols (quercetin and buteine) were selected for the inhibition study. All the catechins inhibited PfENR potently with Ki values in nanomolar range. Among the five compounds studied, EGCG was found to be the best inhibitor. All of them blocked the NADH binding site showing competitive kinetics with respect to NADH and uncompetitive kinetics with crotonoyl-CoA, the substrate analog. Most importantly, the catechins potentiated the inhibition of PfENR by triclosan, a well known PfENR inhibitor. We also report that in the presence of tea catechins triclosan behaves as a slow-tight binding inhibitor of PfENR. The overall inhibition constant of triclosan in the presence of EGCG was calculated to be 2pM which is 50 times better than the earlier reported values with NAD+ (Kapoor, et. al., 2004).

Sink limitations in plants reduce the potential for photosynthesis and yield, particularly under conditions that favour enhanced source activity such as elevated CO2 (EC). Dark respiration, considered catabolic, has rarely been exploited to enhance sink activity in plants. Arabidopsis thaliana L. lines with partially-suppressed mitochondrial pyruvate dehydrogenase (mtPDH) kinase (mtPDHK), a negative post-translational regulator of the mtPDH complex, was shown previously to have both elevated mtPDH complex activity and increased seed weight and oil content at ambient CO2 (AC), suggesting an enhancement of sink activity. The mtPDH links glycolysis with the tricarboxylic acid (TCA) cycle. It was hypothesized that Arabidopsis having suppressed mtPDHK will display their greatest plant productivity at EC through a combined enhancement of source and sink activities. Control and transgenic Arabidopsis having either constitutive or seed-specific expression of antisense mtPDHK were grown at either AC or EC. Expression of mtPDHK and mtPDH complex activity in rosette leaves and reproductive tissues were measured, which required the development of an assay to quantify mtPDH activity. Vegetative and reproductive growth over time, seed oil parameters, and leaf net C exchange were also quantified. A parabolic relationship was found between mtPDHK expression and mtPDH activity, reflecting a role for mtPDH in balancing photosynthetic and respiratory processes. A number of growth and seed oil parameters were improved in transgenic lines, particularly at EC; many of these parameters showed a significant linear or quadratic correlation with mtPDHK expression and mtPDH activity. The proportion of very long chain fatty acids was increased in transgenic lines. Leaf net C exchange was enhanced at AC and EC, and particularly in lines showing repression of mtPDHK. The greatest enhancement in total seed and oil productivity was found for the constitutive lines 104 and 31 at EC (up to 2.8 times). These two lines exhibited a significant increase in inflorescence size, an increase in leaf water use efficiency, the lowest rate of mtPDH complex inactivation by ATP, and an intermediary enhancement of mtPDH complex activity in seeds. Thus, it is concluded that the mtPDH plays a key role in regulating sink and source activities in plants. / Natural Sciences and Engineering Research Council (NSERC) through the Green Crop Networks Research Network; Ontario Graduate Scholarship; Syngenta Graduate Scholarship; Ball Farm Services and Agrico Canada Ltd. Scholarship; Mrs. Fred Ball Scholarship; Arthur D. Latornell Scholarship; Hoskins Scholarship; Robb Travel Grant; Registrars and the Deans Scholarship and travel awards and bursaries from the University of Guelph, and the Ontario Agricultural College.